![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 17, Issue 3, 1075-1084, March 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Institute for Genetics and Center for Molecular Medicine, University of Cologne, D-50674 Cologne, Germany;
The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom;
|| Division of Neuroscience, University of Edinburgh, Edinburgh EH8 9JZ, United Kingdom;
¶ Avenir Team, INSERM U29, INMED, Luminy, 13273 Marseille Cedex 09, France; and
# Center for Biochemistry and Center for Molecular Medicine Cologne, University of Cologne, D-50931 Cologne, Germany
Submitted May 2, 2005;
Revised November 28, 2005;
Accepted December 2, 2005
Monitoring Editor: Jeffrey Brodsky
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
). It is unclear why this region is essential because it does not contain the U box, and it appears to be absent in invertebrate orthologues that ubiquitinate effectively (Koegl et al., 1999; Hatakeyama et al., 2001; Mahoney et al., 2002; Hoppe et al., 2004; Richly et al., 2005). It is important to understand the molecular mechanism of Ube4b because it has a key role in the ubiquitin proteasome system (UPS; Hoppe, 2005), it is neuroprotective in polyglutamine disorders (Matsumoto et al., 2004) and an important candidate gene for neuroblastoma (Krona et al., 2003). Information on the substrates of Ube4b is beginning to emerge (Hoppe et al., 2004; Okumura et al., 2004; Spinette et al., 2004; Richly et al., 2005) but there is much still to learn about its regulation.
In the slow Wallerian degeneration mutant mouse (WldS), 70 amino acids of this essential domain of Ube4b form the N-terminus of a chimeric protein that delays Wallerian degeneration of injured axons in mice and rats by 10-fold (see Figure 1A; Lunn et al., 1989; Mack et al., 2001; Adalbert et al., 2005). The chimeric protein is absent in wild-type mice. This sequence (N70) is fused in WldS protein to the full coding sequence of nicotinamide mononucleotide adenylyltransferase (Nmnat1; Conforti et al., 2000; Emanuelli et al., 2001; Mack et al., 2001), implicating the UPS or NAD+ metabolism in regulating axon degeneration. WldS also delays axon degeneration in a wide range of neurodegenerative disorders and acute retrograde axonal degeneration after spinal injury, indicating that axon degeneration mechanisms are more closely related than previously thought (Wang et al., 2002; Ferri et al., 2003; Samsam et al., 2003; Coleman, 2005; Kerschensteiner et al., 2005; Mi et al., 2005). Surprisingly, WldS in vivo has only been found in nuclei, suggesting that downstream axonal effector(s) mediate its remarkable effect on axon degeneration (Mack et al., 2001; Samsam et al., 2003; Sajadi et al., 2004).
|
; Wang et al., 2005). However, axons in vivo are far longer, have a profoundly different environment, and are protected by full-length WldS for up to 3 wk (Crawford et al., 1995; Adalbert et al., 2005). A contribution of N70 to strengthening the neuroprotective phenotype in vivo is suggested by the fact that transgenic mice overexpressing Nmnat1 show a normal rate of Wallerian degeneration (Coleman and Perry, 2002; Conforti and Coleman, unpublished data). N70 could contribute to neuroprotection by perturbing the UPS, which is important for regulating axon degeneration (Zhai et al., 2003; Macinnis and Campenot, 2005). Alternatively, it could influence intranuclear targeting of Nmnat1, with its intrinsic NAD+ synthesis activity, as WldS protein clusters to discrete intranuclear foci in skeletal muscle (Mack et al., 2001) and in many neuronal subtypes in vivo (Haley et al., unpublished results). The inability of successive investigators to detect any increase in NAD+ level when Nmnat1 is manipulated (Mack et al., 2001; Anderson et al., 2002; Araki et al., 2004) suggests that NAD+ is synthesized at highly localized intranuclear sites or immediately is passed to downstream components within the same complex. Either way, intranuclear targeting should be important.
To understand better the function of N70 in both WldS and Ube4b proteins, we sought N70 binding partners. We identified valosin containing protein (VCP) as a direct binding partner of N70 that becomes targeted to discrete intranuclear foci when WldS protein is present. VCP, one of the AAA family of ATPases associated with a variety of activities, has critical roles in the UPS (Dai and Li, 2001; Jarosch et al., 2002; Wang et al., 2004) and many other cellular roles dictated by interacting proteins (Meyer et al., 2000; Mogk et al., 2004; Wang et al., 2004). It accumulates in neuronal nuclei in a range of neurodegenerative diseases and can influence neurodegeneration both positively and negatively (Hirabayashi et al., 2001; Higashiyama et al., 2002; Mizuno et al., 2003; Watts et al., 2004; Schroder et al., 2005). Its intranuclear roles include interaction with Werner protein to influence the DNA damage response pathway (Indig et al., 2004) and the nuclear import of the T-cell-specific adaptor protein (Marti and King, 2005). Our data suggest the possibility of further intranuclear roles for VCP and an important function for the N70 domain in both wild-type Ube4b and WldS proteins.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
The products were cloned with BamHI/SalI into pGEX5X-1 (Amersham Biosciences, Freiburg, Germany), and with NcoI/SalI into the pGBKT7 vector (BD Biosciences Clontech, Heidelberg, Germany). The WldS protein, N70 and further truncation products were similarly PCR-amplified and cloned using the WldS transgene construct (Mack et al., 2001) as template and appropriate primers based on the WldS cDNA (GenBank AF260924
[GenBank]
). Human Ube4b was amplified from a construct kindly provided by Professor James Mahoney using primers 5'-ATCCCGGAATTCATGGAGGAGCTGAGCGCTGAT-3' and 5'-CCGCCTCGAGTTAGTGATCGCTGTTCTGTTT-3' (EcoRI and XhoI sites for cloning into pGEX5X-1 in bold). cDNA sequence encoding full-length WldS protein, Nmnat-1 or Ube4b amino acids 1–70 (N70) was PCR-amplified from the WldS transgene template (Mack et al., 2001) using the high-fidelity enzyme Pfu (Stratagene, Heidelberg, Germany) and appropriate combinations of the following primers (1 + 2, 4 + 2, and 1 + 3, respectively). 5' restriction enzyme tags, added for cloning purposes, are shown in bold and the first or last three bases of sequence derived from the WldS gene are underlined. A single base change to repress the stop codon of WldS Rev and allow read-through of the C-terminal EGFP is double-underlined: 1) 5'-TAGATCCCAAGCTTAACCTTTCACCATTAAGAGGAAAGCGATG-3'; 2) 5'-GCGGGATCCCGTCCCAGAGTGGAATGGTTGTG-3'; 3)5'-TCCTCCCCGCGGGTCTGCTGCACCTATGGGGGA-3'; and 4) 5'-GACTAGCTAGCATGGACTCATCCAAGAAGACAG-3'.
After cloning of pEGFP-N1 (BD Biosciences), all sequences were verified using the Taq FS BigDye-terminator cycle sequencing method on a ABI 377 prism sequencer and the corresponding ABI software. The sequences were analyzed using the GCG program Wisconsin Package Version 10.2 (Accelrys, San Diego, CA).
Full-length WldS DsRed construct was generated by cloning the HindIII/BamHI insert from pEGFP-N1 (above) into pDsRed2-N1 (BD Biosciences). WldS lacking N16 was generated using the following HindIII and BamHI-tagged primers: 5'-TAGCCCAAGCTTTAGGCCGCCACCATGCTTGCTGGTGGACAGACCT-3'; 5'-GCGGGATCCCGTCCCAGAGTGGAATGGTTG-3'.
Pulldown Assays
C57BL/6J mouse brain homogenates were used in this experiment to avoid competition from endogenous WldS protein. Brains were flash-frozen and homogenized in 50 volumes of 50 mM Tris, pH 9.0, and 1% deoxycholate containing protease inhibitor mix (Sigma, Taufkirchen, Germany). After incubating for 30 min at 37°C, insoluble material was removed by centrifugation (10,000 x g, 30 min) and the supernatant was dialyzed against binding buffer (50 mM Tris, pH 7.4, and 0.1% Triton X-100) overnight at 4°C. Homogenates were incubated overnight at 4°C with GST fusion proteins bound to glutathione Sepharose 4B. The beads were washed four times with phosphate-buffered saline (PBS)/0.1% Triton X-100 and resuspended in 3x standard Laemmli sample buffer. Proteins were separated by SDS-PAGE on 12% gels and analyzed by Western blotting.
MALDI-TOF Mass Spectrometry
Proteins fished by a pulldown assay were eluted with double-concentrated SDS-PAGE sample buffer and separated by SDS-PAGE. From the gel individual Coomassie blue-stained protein bands were excised with a scalpel and destained by washing with 25 mM NH4HCO3/50% acetonitrile. For MALDI-TOF mass spectrometry analysis, the samples were dissolved in 5 µl 0.1% aqueous trifluoroacetic acid. MALDI-MS was carried out in linear mode on a Bruker Reflex IV equipped with a video system (Rheinstetten, Germany), a nitrogen UV laser (Omax = 337 nm), and a HiMass detector. One microliter of the sample solution was placed on the target and 1 µl of a freshly prepared saturated solution of sinapinic acid in acetonitrile/H2O (2:1) with 0.1% trifluoroacetic acid was added. The spot was then recrystallized by addition of another 1 µl acetonitrile/H2O (2:1), which resulted in a fine crystalline matrix. For recording of the spectra an acceleration voltage of 20 kV was used, and the detector voltage was adjusted to 1.9 kV. Approx. 500 single laser shots were summed into an accumulated spectrum. Calibration was carried out using the single and doubly protonated ion signal of bovine serum albumin for external calibration. Identification of the mass fingerprint spectra was performed using the Mascot program available from Matrix Science on the World Wide Web (http://www.matrixscience.com/home.html).
Isolation and Immunoprecipitation of Nuclear Proteins
Six mouse brains (ca. 2.5 g) were each homogenized using a Dounce homogenizer in 40 ml precooled nuclear isolation medium (NIM; 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris/HCl, pH 7.4) supplemented with protease inhibitor cocktail (Sigma). Unbroken cells and connective tissue were removed by filtration. The filtered homogenate was then diluted with an equal volume of ice-cold NIM, centrifuged (10 min, 800 x g) and the pellet, including lipids, was resuspended in 40 ml NIM supplemented with protease inhibitor cocktail. After repeating the centrifugation, the new pellet was resuspended in 8 ml NIM, 2 ml sucrose density barrier (SDB; 2.3 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris/HCl, pH 7.4) solution was added and mixed thoroughly. 10 ml of the suspension was underlayered with 2.3 M sucrose and centrifuged for 1 h at 100,000 x g in a Beckman SW41 Ti rotor (Krefeld, Germany). The pellet, together with remaining lipids, was resuspended in PBS containing 1% Triton X-100 and incubated for 2 h at 4°C with continuous agitation. Samples were then centrifuged at 16,000 x g to generate a supernatant containing solubilized nuclear proteins.
For the subsequent immunoprecipitation, protein lysates were preincubated with 30 µl of protein G agarose (Roche) for 2 h at 4°C under constant agitation. Samples were centrifuged (5 min, 500g) to remove proteins unspecifically bound to the protein G agarose and 3 µl of anti-WldS rabbit serum and another 30 µl of protein G agarose were then added and incubated overnight at 4°C with constant agitation. The precipitated proteins bound to protein G agarose were then repeatedly washed by centrifugation at 500 x g at 4°C for 5 min. The final pellet was resuspended in 1 ml PBS, 1% Triton X-100. Samples were then analyzed by SDS-PAGE and immunoblotting.
In Vitro Binding Assays
GST fusion proteins were purified and coupled to glutathione-Sepharose 4B according to the protocol of the manufacturer (Amersham Biosciences). The pGBKT7 constructs containing the T7 promoter were in vitro transcribed and translated incorporating 35S-methionine using the TNT T7 Reticulocyte Lysate Coupled Transcription/Translation kit from Promega (Promega GmbH, Mannheim, Germany). The obtained proteins were mixed with equal amounts of GST fusion proteins and the binding assay was performed as previously described (Dai et al., 1998). Reactions were analyzed by SDS-PAGE, and the gel was fixed with 10% methanol/10% acetic acid before autoradiography. Purified His-tagged VCP was a kind gift from Dr. Sarah Spinette (Johns Hopkins).
Cell Culture and Transfection
Plasmid DNA was isolated using the endonuclease free plasmid kit (QIAGEN, Hilden, Germany). DNA was transfected using LipofectAMINE 2000 (Invitrogen) into COS-7, PC12, or HeLa cells immediately before differentiation by culturing in 100 ng/µl NGF on a type IV collagen substrate (Sigma). The "TV" PC12 subline, stably transfected with a tet-off inducible C-terminal EGFP-tagged VCP construct (Kobayashi et al., 2002) was grown in 1.0 µg/ml doxycycline (Sigma), which was removed to induce VCP/EGFP expression. Protein location was observed 1–5 d after transfection. Spinal motor neurons from embryonic day 14 (E14) embryonic rats were cultured and electroporated in suspension as previously described (Henderson et al., 1995; Raoul et al., 2002).
Immunocytochemistry
Slices of 100 µm were cut from the cerebellum of WldS mice and fixed in 4% paraformaldehyde (Fisher, Schwerte, Germany). Lumbar spinal cord dorsal root ganglia (DRG) were removed from WldS mice or rats and fixed in 4% paraformaldehyde, and then 20-µm sections cut on a cryostat. Slices were incubated for 2 h at room temperature in serum blocker consisting of 4% bovine serum albumin (Sigma) and 0.5% Triton X-100 (Sigma) in PBS. Wld-18 antibody (Samsam et al., 2003; 1:500 dilution) plus VCP antibody (against amino acids 9–130; BD Biosciences; 1:200 dilution; or mouse monoclonal to VCP (ab11433); AbCam, Cambridge, United Kingdom, 1:500 dilution) were applied simultaneously in serum blocking solution overnight at 4°C. After washing with PBS, slices were incubated overnight at 4°C in a solution containing TRITC-conjugated anti-rabbit (DAKO, Hamburg, Germany; 1:20 dilution in PBS) plus Alexa488-conjugated anti-mouse (Molecular Probes, Leiden, Netherlands; 1:200) secondary antibodies. Slices were then washed in PBS and incubated in To-pro3 (Molecular Probes) for 10 min before mounting in Mowiol/DABCO preparation. Staining was visualized on a laser scanning confocal microscope (Bio-Rad Radiance 2000, Hemel Hempsted, United Kingdom) and Z-series were merged using Lasersharp (Bio-Rad) software.
Cultured cells were fixed for 30 min in 4% paraformaldehyde, permeabilized with Triton-X-100 (0.1%, 5 min), blocked with horse serum (5%, 1 h), and incubated with primary (1 h) and secondary (45 min) antibodies with multiple washes in PBS between each stage.
VCP antibody was used as described above. Anti-ubiquitin polyclonal antibody (DAKO) was used at 1:100 dilution and anti-neurofilament medium chain polyclonal antibody (Chemicon, Hofheim, Germany; Ab1987) was used as 1:1000. Images were taken on a Zeiss LSM 510 META confocal system (Oberkochen, Germany; LSM Software Release 3.2) coupled to a Zeiss Axiovert 200 microscope.
Western Blotting
WldS protein expression was analyzed in mouse cerebella homogenized in five volumes of RIPA buffer, respectively, plus 1x Complete protease inhibitor cocktail (Roche) or in PC12 cells scraped from the dish in a minimal volume of the same buffer. Proteins were separated by SDS-PAGE and semidry blotted onto nitrocellulose (Bio-Rad). Blocking and incubation with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (1:5000; Serotec, Heidelberg, Germany) were performed in PBS plus 0.02% Tween-20 and 5% low fat milk. Proteins were visualized using the ECL detection kit (Amersham Biosciences) according to the manufacturer's instructions.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
; Coleman and Ribchester, 2004), we then tested by Western blotting of WldS and wild-type brain homogenates whether WldS alters turnover of VCP, and thus its steady-state level, and found that it does not (Figure 1F). Thus, VCP directly binds to WldS protein through its N70 domain, and this binding does not significantly alter steady-state level of VCP in brain.
|
; Figure 2). After transient transfection of WldS/EGFP fusion construct into PC12 cells, VCP partially shifted from the widespread nuclear and cytoplasmic distribution that was previously described (Hirabayashi et al., 2001; Kobayashi et al., 2002) and is present in untransfected cells (Figure 2B, arrow) to produce the same pattern of intranuclear foci as WldS (Figure 2B, arrowhead). VCP is an abundant protein (Dai and Li, 2001), so the wider nuclear and cytoplasmic pool remained without any obvious depletion (Figure 2, A–C). VCP was also partially redistributed by WldS in HeLa cells which, like PC12, remained healthy (Supplementary Figures 2 and 4).
|
; Samsam et al., 2003), and VCP colocalization with WldS spots was not evident in vitro (Supplementary Figure 5). Nevertheless, the data are sufficient to conclude that WldS and VCP bind one another under physiological conditions, including in at least some neurons that display the WldS phenotype.
VCP Binds within the First 16 Residues of WldS
We show above that WldS binds to and partially redistributes VCP. To establish that binding causes redistribution, we mapped and then deleted key residues of WldS required for VCP binding. Truncated N70 constructs were expressed as GST-fusion proteins and used to pull down IVTT-expressed VCP. First the N-terminal 23 amino acids were found to be necessary and sufficient for VCP binding, and this was subsequently narrowed to the N-terminal 16 amino acids (N16; Figure 3A). Ube4b lacking this sequence also did not bind VCP. Two WldS/DsRed2 constructs were then made: one full-length and one lacking N16, and transiently transfected into the TV PC12 stable subline that expresses VCP/EGFP fusion protein in a tet-off-inducible manner (a kind gift from Prof. Akira Kakizuka). Full-length WldS/DsRed2 was able to bring about partial redistribution of VCP/EGFP into intranuclear foci in transfected cells (arrowhead; Figure 3, B–E) but removal of N16 containing the VCP binding site prevented this (Figure 3, F–I). Interestingly, the ability of WldS protein to accumulate in intranuclear foci does not require binding to VCP (Figure 3F). Thus, VCP has to bind WldS N16 to be partially redistributed in the manner we describe.
The Sequence Targeting WldS Protein to Intranuclear Foci Is Not Nmnat1
Because VCP binding is not required to target WldS to intranuclear foci, we asked whether this is a property of the Nmnat1 sequence by fusing Nmnat1 to EGFP and transiently transfecting it (Figure 4). Nmnat1, but not N70, was restricted to the nucleus, confirming previous reports that Nmnat1 is a nuclear protein and suggesting that nuclear targeting of WldS protein is due to the putative nuclear localization signal of Nmnat1 (Raffaelli et al., 2002; Araki et al., 2004; Magni et al., 2004). However, unlike WldS/EGFP, intranuclear distribution of Nmnat1/EGFP was homogeneous apart from its exclusion from nucleoli. The fraction of N70/EGFP that entered the nucleus was also homogeneously distributed, suggesting that WldS protein is targeted to intranuclear foci either by a conformational structure involving both N70 and Nmnat1 sequence or by the short unique sequence that separates these two parts of the protein. Cell and nuclear shape indicated that the cells were healthy in all cases. We conclude that Nmnat1 and WldS protein differ in their intranuclear distribution, but as shown above, their differing abilities to bind VCP do not underlie this difference.
|
WldS Also Can Partially Relocalize Ubiquitin inside the Nucleus
VCP binds long ubiquitin chains (Dai and Li, 2001) so one consequence of its partial redistribution by WldS protein could be redistribution of bound polyubiquitinated proteins. This was supported by immunocytochemistry of PC12 cells transiently transfected with WldS/EGFP fusion construct (Figure 5, A–H). As with VCP, the normal distribution of ubiquitin throughout the nucleus and cytoplasm of untransfected cells is joined by a punctate intranuclear pattern in transfected cells, where ubiquitin puncta colocalize with WldS/EGFP. Cell and nuclear shape indicated that the cells were healthy (see also Supplementary Figure 3). The association of ubiquitin epitopes with WldS also depended on the N16 sequence, suggesting it is secondary to VCP binding to WldS (Figure 5, I–P).
|
|
; Richly et al., 2005) and Caenorhabditis elegans (C. Kähler and T. Hoppe, personal communication) so the interaction in these species must have a different molecular basis. The Cdc48 binding site in S. cerevisiae has been mapped (Richly et al., 2005) and mammals do have a sequence homologous to it. However, this sequence appears to be insufficient for VCP binding in human, because N-terminally truncated Ube4b did not bind VCP (Figure 3A). Mammalian Ube4b was previously reported to bind VCP (Meyer et al., 2000; Kaneko et al., 2003) but the interacting sequence was not fully mapped. Our data show that VCP binds to N16, and this appears to be a recent event in evolutionary terms with distantly related higher eukaryotes employing a different mode of interaction.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
A recent report indicates that Nmnat1, but not N70, is sufficient to confer a WldS-like phenotype on neurons in vitro (Araki et al., 2004). However, the relative strengths of the neuroprotective phenotypes induced by Nmnat1 and WldS were not tested and Nmnat1 was not shown to protect in vivo, where WldS robustly protects longer axons for far greater time periods (Mack et al., 2001; Adalbert et al., 2005). Our data show that N70 is not an inert addition to Nmnat1, but instead concentrates VCP, NAD+ synthesis activity and probably its associated ubiquitinated proteins into subnuclear sites, functions that each have potential to influence the strength of the WldS phenotype. Thus, WldS joins a growing list of chimeric proteins whose biological activity amounts to more than the sum of their parts (Fujimoto et al., 1996; Campbell et al., 1997; Blume-Jensen and Hunter, 2001).
Further studies are needed to test whether VCP binding is required for the neuroprotective WldS phenotype, but this possibility was upheld in two important tests. First, WldS and VCP show colocalization in at least some DRG neurons known to express the neuroprotective phenotype, as well as in cerebellum where the phenotype in vivo remains to be tested. Second, colocalization was conserved across species in DRG of WldS rats (Adalbert et al., 2005). The more homogeneous intranuclear distribution of WldS in motor neurons in vivo makes colocalization in this cell type more difficult to test (Mack et al., 2001; Samsam et al., 2003). Cultured motor neurons electroporated with WldS/EGFP show WldS puncta without obvious colocalization of VCP but this does not exclude a role for VCP in the neuroprotective phenotype in vivo (Supplementary Figure 5).
Colocalization studies cannot ultimately answer the question of whether VCP is required for the WldS phenotype. Unfortunately, VCP knockout mice are not available to test the hypothesis and a more complex strategy may be needed. Given the essential role of VCP in endoplasmic reticulum-associated protein degradation (ERAD) and several reports of damaging effects of VCP RNAi, mutation, and deletion in other species or cell lines, it is unlikely that such knockout mice would be viable (Hirabayashi et al., 2001; Kobayashi et al., 2002; Wojcik et al., 2004; Yamanaka et al., 2004). In contrast, WldS alters VCP in a way that leaves all cells and organisms healthy, with no overt harmful effect in the original mutant (Lunn et al., 1989), four lines of transgenic mice (Mack et al., 2001), three of rats (Adalbert et al., 2005) or in double homozygous mice or rats (unpublished observations). This is likely to reflect the confinement of alterations to the nucleus, leaving the essential role of VCP in ERAD unaffected.
Studies in yeast suggest VCP stimulates substrate binding but limits the subsequent growth of a polyubiquitin chain (Richly et al., 2005). However, VCP is dispensable for Ube4b activity when concentrated preparations of protein are used in vitro (Hatakeyama et al., 2001; Mahoney et al., 2002) and in these circumstances removal of the N-terminal 123 amino acids of Ube4b blocks activity for unknown reasons (Mahoney et al., 2002). Our data further highlight the importance of the N-terminus of Ube4b as a regulatory sequence. Many invertebrates lack this sequence but still bind the VCP ortholog Cdc48, so this molecular interaction appears to have changed during evolution.
VCP plays pivotal roles in ERAD, nuclear envelope reconstruction, cell cycle, postmitotic Golgi reassembly, and suppression of apoptosis (Kondo et al., 1997; Dai and Li, 2001; Hetzer et al., 2001; Rabinovich et al., 2002; Wang et al., 2004). Its nuclear functions are equally diverse. First, a nuclear transport role is suggested by its association with adapter proteins Ufd1 and Np14, and by VCP-dependent transport of T-cell-specific adaptor protein into eukaryotic nuclei (Meyer et al., 2000; Marti and King, 2005). Second, VCP controls nucleolar retention of Werner syndrome helicase and its release after DNA damage (Indig et al., 2004). Interestingly, our data fit with the proposal of these authors that other nuclear binding partners regulate VCP distribution and control other pathways. Third, the nucleus has a quality control pathway (Gardner et al., 2005) likely to involve VCP. Interaction and colocalization with expanded polyglutamine and other intranuclear inclusions suggests VCP may be trying to clear misfolded nuclear proteins (Hirabayashi et al., 2001; Doss-Pepe et al., 2003; Mizuno et al., 2003). Finally, VCP regulates the stability of the transcription factor SPT23 (Richly et al., 2005), consistent with the tight regulation of transcription by the UPS (Muratani and Tansey, 2003).
VCP is altered in several neurodegenerative disorders so its interaction with the neuroprotective WldS protein is particularly interesting. VCP missense mutations cause inclusion body myopathy with Paget disease of bone and frontotemporal dementia, a disease characterized by ubiquitin-containing nuclear inclusions and white matter pathology (Watts et al., 2004; Schroder et al., 2005). VCP is present in Lewy-like inclusions in amyotrophic lateral sclerosis, in nigral Lewy neurites in Parkinson's disease (Ishigaki et al., 2004), and in ubiquitin-positive intraneuronal inclusions in motor neuron disease with dementia, ballooned neurons in Creutzfeldt-Jakob disease, and dystrophic neurites of senile plaque in Alzheimer's disease (Mizuno et al., 2003). In polyglutamine disorders such as Huntington's disease and Machado-Joseph disease, specific binding to expanded polyglutamine targets VCP to intranuclear inclusions (Hirabayashi et al., 2001). This interaction appears to influence disease severity, as loss-of-function mutations in the Drosophila VCP ortholog, ter4, dominantly suppress neurodegeneration caused by expanded polyglutamine (Higashiyama et al., 2002).
It is now important to determine whether WldS protein, or N70 can interfere with the effect of VCP in any of these neurodegenerative disorders. Ectopic expression in Drosophila of mammalian Ube4b suppresses polyglutamine disease, and loss of function mutations in VCP have a similar effect (Higashiyama et al., 2002; Matsumoto et al., 2004). It is also now clear that WldS and expanded polyglutamine both bind VCP directly and partially relocalize it to intranuclear foci (Hirabayashi et al., 2001). Taken together, these observations raise the intriguing possibility that N70 and VCP antagonize each other in some circumstances as a result of the binding interaction we describe.
In summary, we report direct interaction between VCP and the N-terminal 16 amino acids of both WldS and Ube4b proteins. This interaction drives focal intranuclear clustering of VCP in WldS neurons, probably together with associated multiubiquitinated proteins, and helps understand the function of an important, evolutionarily recent regulatory sequence in the Ube4b protein. It is now important to determine whether the redistribution of covalently attached Nmnat1 or of bound VCP influences the strength of the WldS phenotype in vivo and to understand how the N-terminal binding of VCP to Ube4b influences this ubiquitin ligase.
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
Abbreviations used: DRG, dorsal root ganglion; ERAD, endoplasmic reticulum-associated protein degradation; IVTT, in vitro transcription and translation; N16, N-terminal 16 amino acids of WldS and Ube4b; N70, N-terminal 70 amino acids of WldS and Ube4b; Nmnat1, nicotinamide mononucleotide adenylyltransferase; Ube4b, ubiquitination factor E4b; Ufd2, ubiquitin fusion degradation protein 2; UPS, ubiquitin proteasome system; VCP, valosin-containing protein; WldS, slow Wallerian degeneration gene or protein.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
These authors contributed equally to this work.
Present address: International University Bremen, D-28759 Bremen, Germany.
Address correspondence to: Michael P. Coleman (michael.coleman{at}bbsrc.ac.uk).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Alonso, M. B. et al. ((2004). ). Identification and characterization of ZFP-57, a novel zinc finger transcription factor in the mammalian peripheral nervous system. J. Biol. Chem. 279, , 25653–25664.
Anderson, R. M., Bitterman, K. J., Wood, J. G., Medvedik, O., Cohen, H., Lin, S. S., Manchester, J. K., Gordon, J. I., and Sinclair, D. A. ((2002). ). Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels. J. Biol. Chem. 277, , 18881–18890.
Araki, T., Sasaki, Y., and Milbrandt, J. ((2004). ). Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science 305, , 1010–1013.
Blume-Jensen, P., and Hunter, T. ((2001). ). Oncogenic kinase signalling. Nature 411, , 355–365.[CrossRef][Medline]
Campbell, R. K., Bergert, E. R., Wang, Y., Morris, J. C., and Moyle, W. R. ((1997). ). Chimeric proteins can exceed the sum of their parts: implications for evolution and protein design. Nat. Biotechnol. 15, , 439–443.[CrossRef][Medline]
Coleman, M. P. ((2005). ). Axon degeneration mechanisms: commonality amid diversity. Nat. Rev. Neurosci. 6, , 889–898.[CrossRef][Medline]
Coleman, M. P., and Perry, V. H. ((2002). ). Axon pathology in neurological disease: a neglected therapeutic target. Trends Neurosci. 25, , 532–537.[CrossRef][Medline]
Coleman, M. P., and Ribchester, R. R. ((2004). ). Programmed axon death, synaptic dysfunction and the ubiquitin proteasome system. Curr. Drug Targets CNS Neurol. Disord. 3, , 227–238.
Conforti, L., Tarlton, A., Mack, T. G., Mi, W., Buckmaster, E. A., Wagner, D., Perry, V. H., and Coleman, M. P. ((2000). ). A Ufd2/D4Cole1e chimeric protein and overexpression of rbp7 in the slow wallerian degeneration (WldS) mouse. Proc. Natl. Acad. Sci. USA 97, , 11377–11382.
Crawford, T. O., Hsieh, S. T., Schryer, B. L., and Glass, J. D. ((1995). ). Prolonged axonal survival in transected nerves of C57BL/Ola mice is independent of age. J. Neurocytol. 24, , 333–340.[CrossRef][Medline]
Dai, R. M., Chen, E., Longo, D. L., Gorbea, C. M., and Li, C. C. ((1998). ). Involvement of valosin-containing protein, an ATPase Co-purified with IkappaBalpha and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IkappaBalpha. J. Biol. Chem. 273, , 3562–3573.
Dai, R. M., and Li, C. C. ((2001). ). Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation. Nat. Cell Biol. 3, , 740–744.[CrossRef][Medline]
Doss-Pepe, E. W., Stenroos, E. S., Johnson, W. G., and Madura, K. ((2003). ). Ataxin-3 interactions with rad23 and valosin-containing protein and its associations with ubiquitin chains and the proteasome are consistent with a role in ubiquitin-mediated proteolysis. Mol. Cell. Biol. 23, , 6469–6483.
Emanuelli, M., Carnevali, F., Saccucci, F., Pierella, F., Amici, A., Raffaelli, N., and Magni, G. ((2001). ). Human NMN adenylyltransferase: molecular cloning, chromosomal localization, tissue mRNA levels, bacterial; Expression, and enzymatic properties. J. Biol. Chem. 276, , 406–412.
Ferri, A., Sanes, J. R., Coleman, M. P., Cunningham, J. M., and Kato, A. C. ((2003). ). Inhibiting axon degeneration and synapse loss attenuates apoptosis and disease progression in a mouse model of motoneuron disease. Curr. Biol. 13, , 669–673.[CrossRef][Medline]
Fujimoto, J., Shiota, M., Iwahara, T., Seki, N., Satoh, H., Mori, S., and Yamamoto, T. ((1996). ). Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5). Proc. Natl. Acad. Sci. USA 93, , 4181–4186.
Gardner, R. G., Nelson, Z. W., and Gottschling, D. E. ((2005). ). Degradation-mediated protein quality control in the nucleus. Cell 120, , 803–815.[CrossRef][Medline]
Hatakeyama, S., Yada, M., Matsumoto, M., Ishida, N., and Nakayama, K. I. ((2001). ). U-Box proteins as a new family of ubiquitin-protein ligases. J. Biol. Chem. 276, , 32111–33120.
Henderson, C. E., Bloch-Gallego, E., and Camu, W. ((1995). ). Purified embryonic motoneurons. In: Nerve Cell Culture: A Practical Approach, ed. J. Cohen and G. Wilkin, London: Oxford University Press, 69–81.
Hetzer, M., Meyer, H. H., Walther, T. C., Bilbao-Cortes, D., Warren, G., and Mattaj, I. W. ((2001). ). Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly. Nat. Cell Biol. 3, , 1086–1091.[CrossRef][Medline]
Higashiyama, H., Hirose, F., Yamaguchi, M., Inoue, Y. H., Fujikake, N., Matsukage, A., and Kakizuka, A. ((2002). ). Identification of ter94, Drosophila VCP, as a modulator of polyglutamine-induced neurodegeneration. Cell Death Differ. 9, , 264–273.[CrossRef][Medline]
Hirabayashi, M. et al. ((2001). ). VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration. Cell Death Differ. 8, , 977–984.[CrossRef][Medline]
Hoppe, T. ((2005). ). Multiubiquitylation by E4 enzymes: `one size' doesn't fit all. Trends Biochem. Sci. 30, , 183–187.[CrossRef][Medline]
Hoppe, T., Cassata, G., Barral, J. M., Springer, W., Hutagalung, A. H., Epstein, H. F., and Baumeister, R. ((2004). ). Regulation of the myosin-directed chaperone UNC-45 by a novel E3/E4-multiubiquitylation complex in C. elegans. Cell 118, , 337–349.[CrossRef][Medline]
Indig, F. E., Partridge, J. J., Kobbe Cv, C., Aladjem, M. I., Latterich, M., and Bohr, V. A. ((2004). ). Werner syndrome protein directly binds to the AAA ATPase p97/VCP in an ATP-dependent fashion. J. Struct. Biol. 146, , 251–259.[CrossRef][Medline]
Ishigaki, S., Hishikawa, N., Niwa, J., Iemura, S., Natsume, T., Hori, S., Kakizuka, A., Tanaka, K., and Sobue, G. ((2004). ). Physical and functional interaction between Dorfin and Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders. J. Biol. Chem. 279, , 51376–51385.
Jarosch, E., Taxis, C., Volkwein, C., Bordallo, J., Finley, D., Wolf, D. H., and Sommer, T. ((2002). ). Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nat. Cell Biol. 4, , 134–139.[CrossRef][Medline]
Kaneko, C., Hatakeyama, S., Matsumoto, M., Yada, M., Nakayama, K., and Nakayama, K. I. ((2003). ). Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a. Biochem. Biophys. Res. Commun. 300, , 297–304.[CrossRef][Medline]
Kerschensteiner, M., Schwab, M. E., Lichtman, J. W., and Misgeld, T. ((2005). ). In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat. Med. 11, , 572–577.[CrossRef][Medline]
Kobayashi, T., Tanaka, K., Inoue, K., and Kakizuka, A. ((2002). ). Functional ATPase activity of p97/valosin-containing protein (VCP) is required for the quality control of endoplasmic reticulum in neuronally differentiated mammalian PC12 cells. J. Biol. Chem. 277, , 47358–47365.
Koegl, M., Hoppe, T., Schlenker, S., Ulrich, H. D., Mayer, T. U., and Jentsch, S. ((1999). ). A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell 96, , 635–644.[CrossRef][Medline]
Kondo, H., Rabouille, C., Newman, R., Levine, T. P., Pappin, D., Freemont, P., and Warren, G. ((1997). ). p47 is a cofactor for p97-mediated membrane fusion. Nature 388, , 75–78.[CrossRef][Medline]
Krona, C., Ejeskar, K., Abel, F., Kogner, P., Bjelke, J., Bjork, E., Sjoberg, R. M., and Martinsson, T. ((2003). ). Screening for gene mutations in a 500 kb neuroblastoma tumor suppressor candidate region in chromosome 1p; mutation and stage-specific expression in UBE4B/UFD2. Oncogene 22, , 2343–2351.[CrossRef][Medline]
Lunn, E. R., Perry, V. H., Brown, M. C., Rosen, H., and Gordon, S. ((1989). ). Absence of Wallerian degeneration does not hinder regeneration in peripheral nerve. Eur. J. Neurosci. 1, , 27–33.[Medline]
Macinnis, B. L., and Campenot, R. B. ((2005). ). Regulation of Wallerian degeneration and nerve growth factor withdrawal-induced pruning of axons of sympathetic neurons by the proteasome and the MEK/Erk pathway. Mol. Cell Neurosci. 28, , 430–439.[CrossRef][Medline]
Mack, T. G. et al. ((2001). ). Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene. Nat. Neurosci. 4, , 1199–1206.
Magni, G., Amici, A., Emanuelli, M., Orsomando, G., Raffaelli, N., and Ruggieri, S. ((2004). ). Structure and function of nicotinamide mononucleotide adenylyltransferase. Curr. Med. Chem. 11, , 873–885.[CrossRef][Medline]
Mahoney, J. A., Odin, J. A., White, S. M., Shaffer, D., Koff, A., Casciola-Rosen, L., and Rosen, A. ((2002). ). The human homologue of the yeast polyubiquitination factor Ufd2p is cleaved by caspase 6 and granzyme B during apoptosis. Biochem. J. 361, , 587–595.[CrossRef][Medline]
Marti, F., and King, P. D. ((2005). ). The p95–100 kDa ligand of the T cell-specific adaptor (TSAd) protein Src-homology-2 (SH2) domain implicated in TSAd nuclear import is p97 Valosin-containing protein (VCP). Immunol. Lett. 97, , 235–243.[CrossRef][Medline]
Matsumoto, M., Yada, M., Hatakeyama, S., Ishimoto, H., Tanimura, T., Tsuji, S., Kakizuka, A., Kitagawa, M., and Nakayama, K. I. ((2004). ). Molecular clearance of ataxin-3 is regulated by a mammalian E4. EMBO J. 23, , 659–669.[CrossRef][Medline]
Meyer, H. H., Shorter, J. G., Seemann, J., Pappin, D., and Warren, G. ((2000). ). A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways. EMBO J. 19, , 2181–2192.[CrossRef][Medline]
Mi, W., Beirowski, B., Gillingwater, T. H., Adalbert, R., Wagner, D., Grumme, D., Osaka, H., Conforti, L., Arnhold, S., Addicks, K., Wada, K., Ribchester, R. R., and Coleman, M. P. ((2005). ). The slow Wallerian degeneration gene, WldS, inhibits axonal spheroid pathology in gracile axonal dystrophy mice. Brain 128, , 405–416.
Mizuno, Y., Hori, S., Kakizuka, A., and Okamoto, K. ((2003). ). Vacuole-creating protein in neurodegenerative diseases in humans. Neurosci. Lett. 343, , 77–80.[CrossRef][Medline]
Mogk, A., Dougan, D., Weibezahn, J., Schlieker, C., Turgay, K., and Bukau, B. ((2004). ). Broad yet high substrate specificity: the challenge of AAA+ proteins. J. Struct. Biol. 146, , 90–98.[CrossRef][Medline]
Muratani, M., and Tansey, W. P. ((2003). ). How the ubiquitin-proteasome system controls transcription. Nat. Rev. Mol. Cell. Biol. 4, , 192–201.[CrossRef][Medline]
Okumura, F., Hatakeyama, S., Matsumoto, M., Kamura, T., and Nakayama, K. I. ((2004). ). Functional regulation of FEZ1 by the U-box-type ubiquitin ligase E4B contributes to neuritogenesis. J. Biol. Chem. 279, , 53533–53543.
Rabinovich, E., Kerem, A., Frohlich, K. U., Diamant, N., and Bar-Nun, S. ((2002). ). AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Mol. Cell. Biol. 22, , 626–634.
Raffaelli, N., Sorci, L., Amici, A., Emanuelli, M., Mazzola, F., and Magni, G. ((2002). ). Identification of a novel human nicotinamide mononucleotide adenylyltransferase. Biochem. Biophys. Res. Commun. 297, , 835–840.[CrossRef][Medline]
Raoul, C., Estevez, A. G., Nishimune, H., Cleveland, D. W., deLapeyriere, O., Henderson, C. E., Haase, G., and Pettmann, B. ((2002). ). Motoneuron death triggered by a specific pathway downstream of Fas potentiation by ALS-linked SOD1 mutations. Neuron 35, , 1067–1083.[CrossRef][Medline]
Richly, H., Rape, M., Braun, S., Rumpf, S., Hoege, C., and Jentsch, S. ((2005). ). A series of ubiquitin binding factors connects CDC48/p97 to substrate multiubiquitylation and proteasomal targeting. Cell 120, , 73–84.[CrossRef][Medline]
Sajadi, A., Schneider, B. L., and Aebischer, P. ((2004). ). Wld(s)-mediated protection of dopaminergic fibers in an animal model of Parkinson disease. Curr. Biol. 14, , 326–330.[CrossRef][Medline]
Samsam, M., Mi, W., Wessig, C., Zielasek, J., Toyka, K. V., Coleman, M. P., and Martini, R. ((2003). ). The Wlds mutation delays robust loss of motor and sensory axons in a genetic model for myelin-related axonopathy. J. Neurosci. 23, , 2833–2839.
Schroder, R., Watts, G. D., Mehta, S. G., Evert, B. O., Broich, P., Fliessbach, K., Pauls, K., Hans, V. H., Kimonis, V., and Thal, D. R. ((2005). ). Mutant valosin-containing protein causes a novel type of frontotemporal dementia. Ann. Neurol. 57, , 457–461.[CrossRef][Medline]
Spinette, S., Lengauer, C., Mahoney, J. A., Jallepalli, P. V., Wang, Z., Casciola-Rosen, L., and Rosen, A. ((2004). ). Ufd2, a novel autoantigen in scleroderma, regulates sister chromatid separation. Cell Cycle 3, , 1638–1644.[Medline]
Wang, J., Zhai, Q., Chen, Y., Lin, E., Gu, W., McBurney, M. W., and He, Z. ((2005). ). A local mechanism mediates NAD-dependent protection of axon degeneration. J. Cell Biol. 170, , 349–355.
Wang, M. S., Davis, A. A., Culver, D. G., and Glass, J. D. ((2002). ). WldS mice are resistant to paclitaxel (taxol) neuropathy. Ann. Neurol. 52, , 442–447.[CrossRef][Medline]
Wang, Q., Song, C., and Li, C. C. ((2004). ). Molecular perspectives on p97-VCP: progress in understanding its structure and diverse biological functions. J. Struct. Biol. 146, , 44–57.[CrossRef][Medline]
Watts, G. D., Wymer, J., Kovach, M. J., Mehta, S. G., Mumm, S., Darvish, D., Pestronk, A., Whyte, M. P., and Kimonis, V. E. ((2004). ). Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nat. Genet. 36, , 377–381.[CrossRef][Medline]
Wojcik, C., Yano, M., and DeMartino, G. N. ((2004). ). RNA interference of valosin-containing protein (VCP/p97) reveals multiple cellular roles linked to ubiquitin/proteasome-dependent proteolysis. J. Cell Sci. 117, , 281–292.
Wu, R., Terry, A. V., Singh, P. B., and Gilbert, D. M. ((2005). ). Differential subnuclear localization and replication timing of histone H3 lysine 9 methylation states. Mol. Biol. Cell 16, , 2872–2881.
Yamanaka, K., Okubo, Y., Suzaki, T., and Ogura, T. ((2004). ). Analysis of the two p97/VCP/Cdc48p proteins of Caenorhabditis elegans and their suppression of polyglutamine-induced protein aggregation. J. Struct. Biol. 146, , 242–250.[CrossRef][Medline]
Zhai, Q., Wang, J., Kim, A., Liu, Q., Watts, R., Hoopfer, E., Mitchison, T., Luo, L., and He, Z. ((2003). ). Involvement of the ubiquitin-proteasome system in the early stages of wallerian degeneration. Neuron 39, , 217–225.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  Y. Sasaki, B. P. S. Vohra, R. H. Baloh, and J. Milbrandt Transgenic Mice Expressing the Nmnat1 Protein Manifest Robust Delay in Axonal Degeneration In Vivo J. Neurosci., May 20, 2009; 29(20): 6526 - 6534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Yahata, S. Yuasa, and T. Araki Nicotinamide Mononucleotide Adenylyltransferase Expression in Mitochondrial Matrix Delays Wallerian Degeneration J. Neurosci., May 13, 2009; 29(19): 6276 - 6284. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Conforti, A. Wilbrey, G. Morreale, L. Janeckova, B. Beirowski, R. Adalbert, F. Mazzola, M. Di Stefano, R. Hartley, E. Babetto, et al. WldS protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice J. Cell Biol., February 23, 2009; 184(4): 491 - 500. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Avery, A. E. Sheehan, K. S. Kerr, J. Wang, and M. R. Freeman WldS requires Nmnat1 enzymatic activity and N16-VCP interactions to suppress Wallerian degeneration J. Cell Biol., February 23, 2009; 184(4): 501 - 513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Beirowski, E. Babetto, J. Gilley, F. Mazzola, L. Conforti, L. Janeckova, G. Magni, R. R. Ribchester, and M. P. Coleman Non-Nuclear WldS Determines Its Neuroprotective Efficacy for Axons and Synapses In Vivo J. Neurosci., January 21, 2009; 29(3): 653 - 668. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Wishart, J. M. Paterson, D. M. Short, S. Meredith, K. A. Robertson, C. Sutherland, M. A. Cousin, M. B. Dutia, and T. H. Gillingwater Differential Proteomics Analysis of Synaptic Proteins Identifies Potential Cellular Targets and Protein Mediators of Synaptic Neuroprotection Conferred by the Slow Wallerian Degeneration (Wlds) Gene Mol. Cell. Proteomics, August 1, 2007; 6(8): 1318 - 1330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Sasaki, T. Araki, and J. Milbrandt Stimulation of Nicotinamide Adenine Dinucleotide Biosynthetic Pathways Delays Axonal Degeneration after Axotomy. J. Neurosci., August 15, 2006; 26(33): 8484 - 8491. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
